hCG is found in pregnancy urine and in urine from some cancer patients in a variety of forms whose concentrations have clinical importance. Recently, concerns about accurate measurement of these forms have been raised because of the finding that hCG with peptide bond cleavages within the beta-subunit is not recognized by commonly used antibodies. Such nicked forms of hCG are biologically inactive or of very low activity. They are present in normal pregnancy urine and to varying extents in the urine of patients with trophoblastic disease. International reference preparations of hCG contain nicked forms of hCG. Previously, it was not possible to separate nicked hormone from the intact form of hCG. This was a serious impediment to producing improved reference standards from natural pregnancy hormone. We now report that a simple hydrophobic purification scheme separates intact hCG from nicked hCG as well as from hCG beta core fragment. This scheme is a modification of the method of Hiyama et al. The order of elution from low to high hydrophobicity is hCG beta core fragment, nicked hCG, and lastly, intact hCG. Nicking of the putative amphipathic helix loop, hCG beta 38-57, apparently renders the hormone significantly less hydrophobic despite the equal molar content of sialic acid. The hCG CR 127 nicked preparation was only 10% as potent as the reference preparation in a heterodimer-directed assay. The nicked-depleted hCG CR 127 was 30% more potent in this assay. Improved hCG reference standards should display similar increases in immunopotency (20-30%) with most antiheterodimeric antibodies and similar increases in bio-potency assays. It should now be possible to make reference preparations of these forms of hCG directly from the raw urine of normal pregnant patients and those with trophoblastic disease.